PROJECT SUMMARY This application is submitted in response to PA-13-194: Mechanisms of alcohol and nicotine co-addiction. Allelic variation in CHRNA5, the gene encoding the ?5 nicotinic acetylcholine receptor (nAChR) subunit, increases vulnerability to alcohol and tobacco dependence. Here, we will use cutting-edge molecular, genetic and behavioral techniques to investigate the role for ?5* nAChRs, particularly those in the medial habenula (MHb)-interpeduncular nucleus (IPN) pathway where ?5* nAChRs are densely expressed, in regulating the motivational properties of alcohol. In Specific Aim I, we will assess alcohol drinking in two lines of mice with deficient ?5* nAChR signaling: ?5 subunit knockout mice and ?humanized? knock-in mice in which the ?5 nAChR subunit gene has been genetically modified to express the major risk allele for tobacco and alcohol dependence in humans. Second, we will use the intracranial self-stimulation (ICSS) procedure to assess the rewarding and aversive effects of alcohol in these lines of mutant mice. We predict that alcohol intake will be increased, and aversive effects of alcohol decreased, in mice with deficient ?5* nAChR signaling. In Specific Aim II, we will investigate the effects of alcohol on the MHb-IPN system. First, we will use optogenetics coupled with electrophysiological recordings to examine the effects of alcohol on excitatory and inhibitory transmission at the MHb-IPN synapse and determine the role for ?5* nAChRs in these effects. Second, we will use rubidium efflux assays to determine the effects of alcohol drinking on the activity of ?5* nAChRs in the MHb-IPN system. We predict that alcohol stimulates excitatory MHb inputs to IPN ? an ?aversion? signal ? and that this effect is attenuated by deficient ?5* nAChR signaling. We further predict that prolonged alcohol intake results in diminished activity of ?5* nAChRs in MHb-IPN system, which may contribute to the development of the alcohol drinking habit. In Specific Aim III, we will investigate the involvement of the MHb-IPN system, and ?5* nAChRs in this system, in regulating alcohol drinking. First, we will use an elegant Cre recombinase-dependent chemogenetics (DREADD) approach to stimulate or inhibit neurons in the MHb-IPN system, or more selectively only those MHb-IPN neurons that express ?5* nAChRs, and examine effects on alcohol drinking. Second, we will use virus-mediated gene transfer to re-express otherwise absent ?5 nAChR subunits in MHb or IPN neurons of the ?5 KO mice and examine the effects on alcohol drinking in these mice. We predict that the MHb-IPN system, and ?5* nAChRs in this system, play a key role in regulating alcohol intake. This innovative program of research may yield novel insights into the mechanisms of alcohol dependence that supports development of entirely new classes of therapeutic agents.